Chromatography

Automated Purification of Native and Recombinant Proteins Using Multidimensional Chromatography

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In traditional sequential chromatography, columns are run as separate entities. The process requires significant hands-on time and constant manual intervention. By contrast, automated chromatography technology provides the same results more efficiently and reliably and frees researchers to focus on other tasks, thereby shortening protein purification times from days to hours. For drug discovery, purifying protein samples is required to generate enough materials for research experiments. But the process is complex and time consuming. It involves repeated single-column purifications, careful analysis,…

February 4, 2016

The Secret Life of Protein A

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Affinity chromatography with protein A has become the foundation for purification of nearly every therapeutic IgG in commercial production. One of the features most responsible for its success has been its compelling simplicity. IgG binds. Contaminants do not. Load, wash, and elute pure IgG. In the real world, however, protein A does not elute pure IgG. It typically contains several hundred to a few thousand parts per million (ppm) contamination by host-cell proteins (HCPs) and other contaminants. Numerous studies demonstrate…

October 15, 2015

Optimization and Scale-Up of HCIC-Based MAb Purification Processes, Part 2

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In multistep schemes, hydrophobic charge-induction chromatography (HCIC) has been shown to contribute effectively to clearance of Chinese hamster ovary (CHO) host-cell proteins (CHOPs), DNA, and viruses. When used for capture chromatography, HCIC can provide better aggregate clearance than protein A sorbents can. Chen et al. enhanced clearance of aggregates, CHOPs, and product- related impurities by controlling HCIC based on both pH and the presence of binding-promoting salt in the wash and elution buffers used (1). Taken together with our findings…

June 16, 2015

Anion-Exchange Chromatographic Clarification: Bringing Simplification, Robustness, and Savings to MAb Purification

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Monoclonal antibodies (MAbs) are the most prominent and successful therapeutic proteins in the pharmaceutical industry. More than 35 MAbs have been approved to treat a range of conditions, and hundreds more are in development (1, 2). Once, the upstream cell culture process was considered the bottleneck to producing high antibody doses required for treatment, but recent advances in cell culture technology have boosted antibody titers to the range of 5–10 g/L (3). That increase in productivity has shifted focus onto…

June 16, 2015

Reagent Clearance Capability of Protein A Chromatography: A Platform Strategy for Elimination of Process Reagent Clearance Testing

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During the manufacturing of monoclonal antibody (MAb) products, many process reagents are used for cell culture and MAb purification to facilitate and control process performance. Process reagents are considered to be process-related impurities, so demonstration of their clearance is required for the chemistry, manufacturing, and controls (CMC) information submission of an investigational new drug (IND) application (1, 2). These reagents may be classified into two categories: generally recognized as safe (GRAS) reagents and potential safety concern (PSC) reagents (3). GRAS…

May 12, 2015

Optimization and Scale-Up of HCIC-Based MAb Purification Processes, Part 1

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Monoclonal antibodies (MAbs) serve important medical needs in cancer treatment as well as that of autoimmune and infectious diseases (1). Antibodies are also widely used in clinical diagnostic assays. They can be coated on solid surfaces to bind specific analytes, conjugated to reporter molecules (either as whole antibodies or fragments) for analyte detection, used in sensitivity panels for lot-release testing, and supplied as positive controls in diagnostic kits (2). Our study evaluates the use of hydrophobic charge-induction chromatography (HCIC) for…

May 12, 2015

Affinity Capture of F(ab’)2 Fragments: Using Twin-Column Countercurrent Chromatography

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Antibody fragments are potent active drug substances (1–4). Because they lack glycosylation, they can be produced using different biological expression systems, including yeast and microbial systems as well as mammalian cells. These molecules are interesting as biopharmaceuticals because they are smaller than full-size antibodies and therefore may penetrate better into different tissues. Antibody fragments are cleared faster in biological systems because they lack the Fc antibody structural region (4). However, fragments may be conjugated to increase their size for improved…

February 6, 2015

Protein A Intermediate Wash Strategies

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Protein A affinity chromatography offers efficient monoclonal antibody (MAb) purification and is used extensively in large-scale MAb production. As is the case with most chromatography media, protein A resins often have some degree of nonspecific binding, which causes host-cell proteins (HCPs) to coelute with a MAb. To reduce nonspecific binding interactions, an intermediate wash step can be performed before product elution. Doing so can improve product purity, extend column lifetime, and potentially eliminate a subsequent polishing step. For large- scale…

February 6, 2015

Advances in Chromatography Automation

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Not long ago, chromatography automation meant strip recorders and peristaltic pumps. Today, few people would consider that to be true automation, and even fewer would settle for binders full of strip-recorder paper reels. Automation is becoming intelligent and in the process is making our workflows smarter. But how close is automation to being as smart as an experienced scientist? Bio-Rad Laboratories spoke with academics, biotechnology R&D scientists, and industrial process engineers about the evolution of chromatography automation — where it…

January 13, 2015

Cost Estimation for Protein A Chromatography: An In Silico Approach to MAb Purification Strategy

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Monoclonal antibody (MAb) production has adopted an accepted technology platform for downstream processing (1). The need for more economic processes has been addressed by increasing MAb titers in fermentation and aiming toward greater bioreactor volumes to increase productivity. Consequently, cost pressures are now passed on to downstream process groups. Membrane and chromatography resin savings are more important for MAb processes than ever before, with highly productive cell cultures generating large volumes of process fluid to purify (2). Traditionally, protein A…

October 16, 2014